CN106999460B

CN106999460B - Palmitoleic acid for inhibiting sea lice from adhering to fish

Info

Publication number: CN106999460B
Application number: CN201580056189.9A
Authority: CN
Inventors: 帕特里克·帕格特
Original assignee: Institut de Recherche en Semiochimie et Ethologie Appliquee
Current assignee: Institut de Recherche en Semiochimie et Ethologie Appliquee
Priority date: 2014-08-12
Filing date: 2015-08-07
Publication date: 2021-07-09
Anticipated expiration: 2035-08-07
Also published as: US11020364B2; EP3179997B1; JP2017530944A; CA2957363A1; AU2015303009A1; NZ728811A; CN106999460A; EP3179997A1; US20170231939A1; PE20170934A1; JP6676619B2; AR101474A1; DK3179997T3; DK201770077A1; CA2957363C; AU2015303009B2; WO2016024168A1; CL2017000302A1; MA40567A; DK179642B1

Abstract

A semiochemical composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof and/or mixtures thereof and an acceptable vehicle is described. The invention also describes a method of treating sea lice comprising administering to fish in need of such treatment a semiochemical composition comprising a sea lice attachment inhibiting semiochemical comprising a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof and/or mixtures thereof and an acceptable vehicle.

Description

Palmitoleic acid for inhibiting sea lice from adhering to fish

Technical Field

The present invention relates to a semiochemical composition comprising a sea lice copepodites (copepodites) attachment inhibiting semiochemical comprising a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof and/or mixtures thereof and an acceptable vehicle. In addition, the present invention is directed to a method of treating sea lice comprising administering to fish in need of such treatment a semiochemical composition comprising a sea lice attachment inhibiting semiochemical comprising a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof and/or mixtures thereof and an acceptable vehicle.

Background

Sea lice are considered to be one of the most important causes of hygiene and economic losses in fishery production (Johnson et al, 2004). In Atlantic salmon (Atlantic salmon/Salmo salar) and rainbow trout (rainbowtrout/Oncorhynchus mykiss), Lepeoptheirus salmonis (Lepeoptheirus salmonis) and other fish lice copepods (caligid copepods) were identified as major obstacles to the development of salmon (salmonid fish) farming, which resulted in authorities restricting the establishment of new farms as long as no satisfactory treatment was available (Stewart et al, 2004).

The common and most important species, louse salmonides salmonis, now appearing to be the most common, exhibit a complex life cycle comprising distinct life stages. Of these, the copepodites stage is of particular interest because it is the time of infestation (Pike and Wadsworth, 1999). As the first larval stage (nauplius I and nauplius II), copepodites (copepods) do not have a functional digestive tract, but use energy obtained from the breakdown of its adipose tissue. Its life appears to be limited to 2 weeks during which time it must be attached to the host by the corner hook. The copepods act as the most planktonic organisms and move passively with the river even though they have the ability to exhibit some rate of movement (which allows them to catch and attach to the host). Literature reports that contact with the host is facilitated by burst swim (burst swim) in response to linear water acceleration (frequency of 3 ± 12Hz, which lasts 1 ± 3 seconds) (Heuch & Karlsen, 1997). This behavior may result from the strategy of avoiding predators observed in free-ranging copepods (Heuch & Karlsen, 1997). The high frequency of first infestation on the fins can be considered as another argument that supports the following hypothesis: the copepods are activated by small scale water movements generated around them (Tully et al, 1993 a). Some authors (Anon, 1993) have suggested that there may be an important passive infestation by the gill cavity, attached larvae (chalimus) migrating to the body surface after metamorphosis. Recent studies have shown that this theory may be caused by human factors during experimental infestation. A low frequency of gill infestation in reports on natural large-scale infestation should be considered as a strong argument against this hypothesis (Tully et al, 1993b and 2002).

After hooking on the fish, the copepodites seem to use chemosensory information. Chemoreceptors for antennae have been described, which may explain the departure and return of copepods attached to inappropriate hosts (non-salmonids) to the river (Bron, 1993).

Various studies have described the role of semiochemicals in the invasive behaviour of the species Lepeopthheirus. Prior to any study that made it possible to identify some chemical compositions, different observations highlighted the possibility of participation of chemical information. The antennal of the copepodites carries the sensory trap and it is also responsible for hooking on the fish (Bon, 1993). As previously mentioned, when copepods are attached to non-salmonids, they tend to unhook and this distinction appears to be likely due to chemical information. The presence of chemical signals released through fish skin is not surprising, since it has been demonstrated that skin mucus comprises various lipid compounds, which are well known to be involved in chemical communication between various species (Lewis, 1970).

However, the most important literature is obtained due to evidence of the attractive effect of "fish conditioned water" obtained by sampling water in which fish have swim (Ingvarsd Louttir et al, 2002; Bailey & Mouerd, 2003; Pino-Marambio et al, 2007& 2008). Some exact compounds have been identified and, between them, isophorone and 1-octen-3-ol appear to be selectively detected by an antenna. Unfortunately, most published data on adult sea lice that has been obtained is not directed to the life cycle of infestation by parasites.

Some other data suggest that stress affects the vulnerability of salmonids (salmonids) to attack by sea lice infestation (Tully et al, 1993a & b; MacKinnon, 1998; Mustafa & MacKinnon, 1999). This stress may involve various stimuli, of which silvering (Barton et al, 1985) and social stress (social stress) appear to be crucial in species that cannot be considered as truly domesticated (Sloman et al, 2001; Gilmour et al, 2005). An increase in plasma cortisol may act by lowering the concentration of antibodies, particularly IgM immunoglobulins, in the skin mucus (Magnado Louvtir, 1998; Hou et al, 1999). These antibodies appear to play a role in immunity against infestation, which may affect the attachment of hair carried by attached larvae (Tully et al, 2002).

Fish farming has resulted in fish escaping from farms and merging into wild salmon populations. This has an impact on disease transmission and food competition in wild fish populations. Farmed fish have many altered characteristics compared to wild fish. One such feature is that farmed fish are more aggressive than their wild fish (Johnson & Abrahams, 1991). Another feature is that the offspring of farmed fish grow faster than wild fish (Fleming et al 2000). The number of hosts of salmon lice has been multiplied by fish farming.

There are two sea lice, the genus callouse (lepeoptheirus salmonis) and the genus louse (Caligus elongatus). They can be identified by their brown horseshoe shaped housing. They attach firmly to fish and are damaged by eating the scale (scale), cell tissue, blood and mucous membranes. The fish immune system is weakened, leading to the possibility of secondary infections and fish death. Salmon smolt (smolts) with more than 10-15 salmon lice are sick and are unlikely to survive their journey through the sea before returning to the river for spawning. Low non-lethal infestation of sea lice can cause stress in fish, resulting in salt imbalance, decreased immunity and increased infection (Tully et al, 2002). Generally, 12-15 sea lice can kill one wild salmon.

Salmon lice have basic treatments, one being biological and the other being chemical. Using a humped head fish, which is a marine fish of the family humped head fish family (family), the ectoparasite salmon louse can be picked up and eaten, which is a biological method. For this purpose, Goldfish (goldsinny wrasee), Goldfish (Ctenolabrasus), Goldfish (Ballam wrasee), Belleville (Labrus bergylta Ascarius), Goldfish (corkwing wrasee), Goldfish (Symphoduseleiops (L.), rock cook fish, Small-bore Pseudosciaenopsis ((Centrolabrus exoluteus) (L.), Goldfish (cuckoo wrasee), Blodfish (Labrus bimaculatus L.), and Pseudosciaenopsis parviflora (scale-rayed) (Acronusratronus parviensis) are used in the aquaculture industry.

The chemical method involves the use of a pediculicide bath or pellets in fish feed. Bath treatments used against salmon lice include: organic phosphates such as dichlorvos, trichlorfon and azamethiphos; pyrethroids such as cypermethrin and deltamethrin; pyrethrum extract and hydrogen peroxide. Feed therapy includes avermectins such as emamectin benzoate, diflubenzuron, teflubenzuron, cypermethrin, cis-cypermethrin and ivermectin. Typically, the addition of SLICE (emamectin benzoate) lasts 8 weeks.

However, resistance to bath and fish feed treatments becomes apparent over time, resulting in inefficient treatments, toxicity to non-target organisms, stress to fish and are expensive. In many cases, a drug holiday (dry period) of treatment is required and re-infestation often occurs, particularly in farmed salmon, where regular parasite treatment puts continuous pressure on the development of resistance.

US patent 7,879,809 describes the use of spinosad or a physiologically acceptable derivative or salt thereof to control ectoparasite infestations in aquaculture fish. Spinosyns are known to be broad spectrum organic pesticides.

US patent 6,982,285 describes an injectable solution with the active ingredient 1- [ 4-chloro-3- (3-chloro-5-trifluoromethyl-2-pyridyloxy) phenyl ] -3- (2, 6-difluorobenzoyl) urea for the control of fish parasites.

Although the use of drugs and chemicals for the treatment of fish parasites such as sea lice is known in the art, they are often environmentally unfriendly. Drug resistance occurs in most cases, resulting in lack of therapeutic efficacy and increased mortality in salmon and other farmed fish. Therefore, other solutions to sea lice infestation are needed.

Semiochemicals are chemical substances emitted by plants or animals that can elicit a behavioral or physiological response in another organism. When a semiochemical affects an individual of the same species, it is called a pheromone (pheromone). When a semiochemical affects individuals of different species, it is known as an allelochemical (allochemochemical).

Those chemical signals involved in interspecies communication are classified as generic for allelochemical signals. Xenobiotic signals are typically divided into two subgroups and their function affects the relationship between the signal releaser and the message recipient. When there is a chemical signal emitted that is directed to favouring the releaser, this subgroup is called allomones (allomones). According to this definition, a heterologous pheromone (allone) is a hormone or substance produced by one species that may have an effect on another species, particularly an effect that is beneficial for the release of the species. For example, attractive heterologous pheromones released by specific flowers may attract a variety of insects that can pollinate these flowers.

Conversely, when the chemical signal released is directed to the benefit of the recipient, then this subgroup is called the kairomone (kairomone; kairomone). By definition, a kairomone is a pheromone or substance that may attract other species, sometimes even natural enemies. Interspecies hormones are sometimes associated with the localization of parasites to a particular host. For example, lactic acid released through human skin is a known interspecific hormone for many mosquito families (Culicideae). Heterologous pheromones and interspecies hormones are natural substances whose degradation does not cause harm to the end user. These chemicals also do not elicit immunity and are safe.

Accordingly, there is a need in the art to provide an environmentally friendly composition and method for inhibiting sea lice attachment to fish that can be easily administered and is effective without harming or stressing the fish or affecting the aquatic environment.

The above needs and other objects are met by the present invention as evidenced by the summary, the description of the preferred embodiments and the claims.

Disclosure of Invention

The present invention relates to a semiochemical composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof that detach sea lice from fish and an acceptable vehicle.

In another aspect, the present invention is directed to a semiochemical composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising between 0.1ppm and about 10ppm of a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof and a nontoxic filler and/or enhancer composition and an acceptable vehicle.

In another aspect, the present invention is directed to a semiochemical composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising between 0.6ppm and about 6ppm of a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof and an acceptable vehicle.

An acceptable excipient as described herein is a pharmaceutically acceptable excipient or a veterinarily acceptable excipient.

As described herein, the sea lice copepodites attachment inhibiting semiochemical compositions comprising synthetic palmitoleic acid may further comprise a non-toxic filler or enhancer composition. The non-toxic filler is selected from the group consisting of: fatty acids, alcohols, amines, squalene, glycerol and mixtures thereof, and enhancer compositions containing amines and fatty acids from indole derivatives, esters of these amines and fatty acids, ketones, acetone, alcohols or sterols.

In another aspect, the semiochemical compositions described herein that inhibit sea lice copepodites attachment are ester, alcohol, ketone, amide, ether, aldehyde or sterol derivatives of synthetic palmitoleic acid, salts, isomers and/or structural analogs thereof that maintain their semiochemical capabilities to detach sea lice from fish, and/or mixtures thereof, and acceptable excipients.

The semiochemical compositions may be in the form of powders, tablets, pills, capsules, granules, dry tablets or other forms suitable for use. It may also be in the form of a sustained release formulation: placed in micelles, liposomes, nanoparticles, microparticles, or microencapsulated. The semiochemical compositions may also be lyophilized.

Another aspect of the invention is a solution comprising a composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising a synthesized palmitoleic acid, salts thereof, isomers thereof and/or structural analogs thereof that retain their semiochemical capabilities and/or mixtures thereof. The solution may be formulated with an acceptable excipient, such as a pharmaceutically acceptable excipient or a pharmaceutically acceptable excipient. The solution may also contain a non-toxic filler as described herein, or a reinforcing agent composition as described herein.

Another aspect of the present invention is that the solution comprises a sea lice copepodites attachment inhibiting semiochemical composition comprising between about 0.1ppm and about 10ppm of a synthesized palmitoleic acid, salts thereof, isomers thereof and/or structural analogs thereof that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof. The solution may be formulated with an acceptable excipient such as a pharmaceutically acceptable excipient or a veterinarily acceptable excipient. The solution may also contain a non-toxic filler as described herein, or a reinforcing agent composition as described herein.

Another aspect of the present invention is that the solution comprises a sea lice copepodites attachment inhibiting semiochemical composition comprising between about 0.6ppm and about 6ppm of a synthesized palmitoleic acid, salts thereof, isomers thereof and/or structural analogs thereof that maintain their semiochemical capabilities and/or mixtures thereof. The solution may be formulated with an acceptable excipient such as a pharmaceutically acceptable excipient or a veterinarily acceptable excipient. The solution may also contain a non-toxic filler as described herein, or a reinforcing agent composition as described herein.

The solution may be in the form of a spray, aerosol, emulsion, suspension, drops, may be placed in an underwater diffuser or in a slow release matrix. It can be added to the water in which the fish reside, or placed in the fish food. It can also be administered to fish orally or by injection.

Another aspect of the invention is a method of repelling sea lice from fish, said method comprising administering to the fish a semiochemical composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof that maintain their semiochemical capabilities to repel sea lice from fish and/or mixtures thereof and an acceptable vehicle.

Another aspect of the invention is a method of repelling sea lice from fish, said method comprising administering to the fish a semiochemical composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising between 0.1ppm and 10ppm of a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs that maintain their semiochemical capabilities to repel sea lice from fish and mixtures thereof and an acceptable vehicle.

Another aspect of the invention is a method of repelling sea lice from fish, said method comprising administering to the fish a semiochemical composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising between 0.6ppm and 6ppm of a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs that maintain their semiochemical capabilities to repel sea lice from fish and mixtures thereof and an acceptable vehicle.

Another aspect of the invention is a semiochemical composition comprising a synthetic palmitoleic acid, salts, derivatives, isomers and/or structural analogs thereof that maintain the semiochemical capabilities thereof to detach sea lice from fish and mixtures thereof and acceptable excipients for use in inhibiting the attachment of sea lice to fish.

Another aspect of the invention is a semiochemical composition comprising between about 0.1ppm and about 10ppm of a synthetic palmitoleic acid, salts, derivatives, isomers and/or structural analogs thereof that maintain the semiochemical capabilities thereof to detach sea lice from fish and mixtures thereof and acceptable excipients for use in inhibiting adhesion of sea lice to fish.

Another aspect of the invention is a semiochemical composition comprising between about 0.6ppm and about 6ppm of a synthetic palmitoleic acid, salts, derivatives, isomers and/or structural analogs thereof that maintain the semiochemical capabilities thereof to detach sea lice from fish and mixtures thereof and acceptable excipients for use in inhibiting adhesion of sea lice to fish.

Drawings

Figure 1 is a photograph of sea lice on juvenile salmon.

FIG. 2 is a schematic diagram showing the Smiley Chamber test setup, which is a test adapted from a conventional olfactometer.

FIG. 3 is a photograph of an original Smiley Chamber test.

Fig. 4 is a photograph of an olfactometer (olfactory) used in the experiment.

Fig. 5 is a photograph of counted copepods after filtering the effluent of the olfactometer.

FIG. 6 is a photograph at the beginning of the infestation test. Smolt were placed in a water tank where they were kept in 3ppm of a semiochemical product containing synthetic palmitoleic acid or placebo (placebo).

Fig. 7 is a photograph showing the harvested copepodites by rubbing the fish with a metal spoon to remove all the copepodites that may be attached to the fish.

Figure 8 is a box plot showing the results of control salmon and control cod compared to treated salmon in example 6.

Fig. 9 is a graph showing the number of copepodites in the control group of example 6.

Fig. 10 is a graph showing the number of copepodites in the treated salmon group of example 6.

Fig. 11 is a graph showing the number of copepodites in the control cod group of example 6.

Detailed Description

Description of the preferred embodiments

As used herein, "fish" includes any member of the following: phylum Chordata (Phylum Chordata) Vertebrate subgenus (Sub Phylum vertibrate) and class amantada (Superclasses Agnatha), class chondrocytozoa (chrondrichties) and class sclerostina (ostochhties), class: actinopterygii (Actinopterygii), order: salmoniformes (Salmoniformes) or Perciformes (Perciformes) or Siluriformes (Siluriformes). For example, but not limited to, the following fish species may be mentioned as encompassed by the present invention: catfish, carp, trout, salmon, sea bass (sea bass), sea bream, whitefish (whitefish), redspot salmon (char), fennel (graining), and bass.

The catfish encompassed by the present invention includes catfish such as catfish (channel catfish), catfish blue (blue catfish), catfish flathead (flathead catfish), catfish yellow (yellow catfish), etc. The carp includes Cyprinus Carpio (common carp), Cyprinus Carpio, and Cyprinus Carpio. The trout comprises: oncorhynchus mykiss (Green Back Cutthroat trout), Rio Grand Oncorhynchus mykiss, Oncorhynchus mykiss (Brook trout), Hybrid Cut-Bow trout, Oncorhynchus mykiss, Palomino Oncorhynchus mykiss, and the like. Salmon include knook salmon (Chinook salon), silver salmon (Coho salon), red salmon (Sockeye salon), dog salmon (Chum salon), Pink salmon (Pink salon), Atlantic salmon, Steelhead salmon (Steelhead salon), and the like. Sea bass (sea bass) includes banked sea bass, chile sea bass (childsea bass), Black sea bass (Black sea bass), Rock sea bass (Rock sea bass), spot sea bass (spot sea bass), and the like. The sea bream includes black sea bream, Red sea bream (Red sea bream). The whitefish includes normal whitefish, lake whitefish (lake whitefish), atlantic whitefish, columba (round whitefish), panwhite fish, northern salmon (Inconnu), Pacific whitefish, Lianhai whitefish, Pagrus major (white steebras), and the like. Redspot salmon includes lambs (Dolly Varden) redspot salmon, Atlantic redspot salmon, Wisconsin ivory redspot salmon, and the like. The Pimpinella anisum comprises Pimpinella anisum and Pimpinella anisum. The weever comprises: pan perch, European perch (European perch), Barkhaus perch (Balkhash perch), Yellow perch (Yellow perch), gold perch (Golden perch), Silver perch (Silver perch), Japanese perch (spread perch), White perch (White perch), and the like.

"stress of fish" refers to physical, chemical or mental discomfort resulting in the release of stress-related hormones or in a particular physiological response. Stress causes physical reactions such as increased heart rate, blood pressure, elevated blood glucose, and the release of cortisol levels. It contains elevated cortisol levels above 100 ng/ml. Increased cortisol levels can lead to myocardial remodeling in salmonids (Johansen et al, The Journal of Experimental Biology (2011)214, 1313-. There are different types of stress in fish, including confinement stress (confinement stress), handling stress (handling stress), sorting stress (sorting stress), grading stress (grading stress), and transport stress (transportation stress). Stress may help to reduce the resistance of the fish, leading to spread of disease and parasitic infestation. It also has an effect on the feeding behaviour, growth and competitive capacity of fish (Gregory et al, physical and biological biology (1999)72 (3): 286-.

"aquaculture" as used herein refers to the science, technology and commerce of growing fish under controlled conditions.

By "active ingredient" is meant a molecule that confers its therapeutic properties as a sea lice copepodites attachment inhibiting semiochemical comprising synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof that retain their semiochemical capabilities and mixtures thereof.

The term "sea lice" as used in the present specification includes any copepod (copepod) of the family carpoideae (Caligidae) of the order siphonostomatoidea (siphenostomada), which includes the genera lepeophtis (lepeophheirus) and pediculosis (Caligus). Examples thereof include: lepeophtheirus salmonis (Lepeophtheirus salmonis), Lepeophtheirus peloralis, Lepeophtheirus thompsonii, Lepeophtheirus europaensis, Caligius elonggatus, Caligius orientalis, Caligius teres, Caligius fish lice (Caligius rogerprocessi), Caligius punctatus, Caligius epididacticus, sea lice (Caligius clemensi), and the like. Sea lice were found in different waters. Thus, for example, the salmon scab lice affect Atlantic salmon in the colder water regions of the northern hemisphere. It also infects Japanese salmonids (salmonidsin Japan). Orientalis is also found in rainbow trout in japan. Elongatus is the most prevalent species in the water areas of the united kingdom, c.ters and c.rogercreatseyi are the most prevalent species in the water areas of chile, c.epidemocus, c.puncutata and c.orientalis are the most prevalent species in the water areas of asia, l.peptoralis occurs in the northeast atlantic ocean, the sea of polo and the white sea, and c.elongatus occurs in the southern hemisphere (particularly in australia).

As used herein, "copepodites" refers to any of a wide variety of very small crustaceans of the Copepoda subclass (subclass Copepoda) having a slender body and a bifurcated tail. Unlike most crustaceans, copepods (copepods) lack the shell of the back and do not have compound eyes. Copepods are abundant in both saltwater and freshwater and are an important food source for many aquatic animals. Copepods include daphnia.

The term "cop" as used herein is an abbreviation for "copepodite".

As used herein, "semiochemical" refers to a chemical substance emitted by a plant or animal that is capable of eliciting a behavioral or physiological response in another organism. When a semiochemical affects an individual of the same species, it is called a pheromone. When a semiochemical affects individuals of different species, it is known as a xenobiotic.

By "heterologous pheromone" is meant a semiochemical produced by one organism that may elicit a response in an organism of another species. It produces a favorable response to the releaser. For example, some plants produce heterologous pheromones that repel insects and prevent them from feeding.

As used herein, "interspecific hormone (kairomone)" is a chemical messenger (messenger) released by an organism of one species that is beneficial to or affects an organism of another species. One example of a xenobiotic is a floral scent that attracts or repels animals.

As used herein, "palmitoleic acid" includes cis-9-hexadecenoic acid and has the formula C₁₆H₃₀O₂. Palmitoleic acid (Palmitoleic acid) is also known as scyloleic acid (zoomatic acid), Palmitoleic acid (palmitonolic acid), (9Z) -hexadecenoic acid, (Z) -hexadec-9-enoic acid, (9Z) -hexadec-9-enoic acid, cis-delta (9) -hexadecenoic acid, 16: ln-7 or 16: 1^Δ9。

By "synthetic" is meant that palmitoleic acid is chemically or enzymatically prepared, and not isolated from nature.

As used herein, "derivatives" include esters, alcohols, ketones, amides, ethers, aldehydes, and sterols of synthetic palmitoleic acid, their derivatives, their salts, their isomers, and/or their structural analogs, and/or mixtures thereof. These synthetic palmitoleic acid derivatives may replace one or more semiochemicals in the compositions as described herein and have the same effect.

Derivatives of fatty acids can be synthesized by methods known in the art. For example, direct esterification of fatty acids with alcohols catalyzed by acid catalysts produces fatty acid esters and water. Fatty aldehydes can be converted to fatty alcohols by hydrogenation. The fatty acid amide may be prepared as follows: esters of fatty acids and lower alcohols are reacted with ammonia or mono-or dialkyl methyl or ethyl amines under anhydrous conditions, and then the lower alcohols are removed from the reaction.

"isomers" include structural and stereoisomeric forms. Structural isomers (structural isomers) are isomers having the same constituent atoms but arranged differently from each other. Examples of structural isomers are propanol and isopropanol. The stereoisomers comprise the same atoms linked in the same way on the molecule and differ from each other only in the spatial arrangement of the atoms or groups of atoms. Examples of the spatial isomers are glucose (glucose) and dextrose (dextrose). Examples of palmitoleic acid isomers include: 11-cis-hexadecenoic acid and 9-cis, 12-cis hexadecanoic acid, and trans-9-hexadecenoic acid.

"structural mimetics" refers to a group of compounds that are similar to each other in structure, but differ from each other in specific composition. Structural mimetics can differ in more than one atom, functional group, or substructure (which is substituted with other atoms, functional groups of a substructure). Examples thereof include: 2-methoxy-5-hexadecenoic acid, halogenated palmitoleic acids such as omega-fluorinated palmitoleic acid, nitrated palmitoleic acid, chlorinated palmitoleic acid, omega-hydroxynonenoic diacid, and the like.

The term "mixture" as used in this specification includes synthetic palmitoleic acid and salts, derivatives, isomers and/or structural analogues thereof, which retain the activity of inhibiting sea lice attachment to fish. For example, the mixture may comprise synthetic palmitoleic acid and isomers of synthetic palmitoleic acid, or they may comprise structural analogs of synthetic palmitoleic acid and derivatives of synthetic palmitoleic acid.

The term "solution" refers to a liquid in which a solid or oil is dispersed in a liquid by dissolution or suspension.

By "acceptable excipient" is meant any pharmaceutically or veterinary excipient that does not interfere with the activity of the sea lice copepodites attachment inhibiting semiochemical compositions comprising synthesized palmitoleic acid, salts, isomers and/or structural analogs thereof, and/or mixtures thereof, and that is non-toxic to the fish to which it is administered.

By "enhancer composition" is meant an active composition that is species specific in fish and which can be used to enhance or act synergistically with a basic semiochemical composition as described herein to enhance the effectiveness of a basic semiochemical composition as described herein in fish.

"administering" means either applying a sea lice copepodites attachment inhibiting semiochemical composition as described herein comprising a synthetic palmitoleic acid to the aqueous environment of a fish or applying a sea lice copepodites attachment inhibiting semiochemical composition as described herein comprising a synthetic palmitoleic acid to a bait for ingestion or applying a sea lice copepodites attachment inhibiting semiochemical composition as described herein directly on or within a fish. Thus, oral, injection, topical administration to fish, and placement of the semiochemical composition in the environment of fish are also contemplated by the present invention.

"Environment" refers to the environment surrounding the fish.

By "consisting essentially of … …," it is meant that the semiochemical compositions used to inhibit the attachment of sea lice copepodites have an active ingredient that synthesizes palmitoleic acid, but may include other compounds that do not affect the semiochemical properties of the active ingredient.

The present invention relates to a semiochemical composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof and an acceptable vehicle.

As described herein, a semiochemical composition comprises a sea lice copepodites attachment inhibiting semiochemical comprising between about 0.1ppm to about 10ppm, or between about 0.6ppm to about 6ppm, or between about 1ppm to about 5ppm, or between about 0.05ppm to about 20ppm of a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof and an acceptable vehicle.

In another aspect, the present invention is directed to a semiochemical composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising between about 0.1ppm to about 10ppm, or between about 0.6ppm to about 6ppm, or between about 1ppm to about 5ppm, or between about 0.05ppm to about 20ppm of a salt of a synthesized palmitoleic acid, a derivative of a synthesized palmitoleic acid, an isomer of a synthesized palmitoleic acid and/or a structural analog of a synthesized palmitoleic acid that maintains the semiochemical capabilities to detach sea lice from fish and/or a non-toxic filler or enhancer composition and an acceptable vehicle.

In another aspect, the present invention is directed to a composition comprising a sea lice copepodites attachment inhibiting semiochemical comprising between about 0.6ppm and about 6ppm of a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof and an acceptable vehicle.

An acceptable excipient is a pharmaceutically acceptable excipient or a veterinarily acceptable excipient. It includes solvents, dispersion media, absorption retarders, and the like. Such pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, 21 st edition (2005). Acceptable excipients may be, for example, glycol ethers or physiological saline. Acceptable excipients will vary with the manner in which the semiochemical composition is formulated. It may be added during formulation to a sea lice copepodites attachment inhibiting semiochemical composition comprising a synthesized palmitoleic acid, or salts, derivatives, isomers and/or structural analogs thereof that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof.

Pharmaceutically acceptable salts of the semiochemical compositions described herein include those which are organic or inorganic salts of synthetic palmitoleic acid. These are known and described in the physicians' Desk Reference, merck index and the pharmacological basis of goodman + gilman therapeutics. Pharmaceutically acceptable salts are, for example, sodium, potassium, ammonium, calcium and magnesium salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and the like, or with organic acids such as oxalic acid, fumaric acid, tartaric acid, malonic acid, acetic acid, citric acid, benzoic acid and the like.

The sea lice copepodites attachment inhibiting semiochemical compositions described herein may further comprise a non-toxic filler or enhancer composition. The non-toxic filler is selected from the group consisting of: fatty acids, alcohols, amines, squalene, glycerol and mixtures thereof, and the enhancer composition comprises: amines and fatty acids from indole derivatives, esters of these amines and fatty acids, ketones, acetone, alcohols or sterols.

In another aspect, the semiochemical compositions described herein that inhibit sea lice copepodites attachment are ester, alcohol, ketone, amide, ether, aldehyde or sterol derivatives of synthetic palmitoleic acid, salts, isomers and/or structural analogs thereof that maintain the semiochemical capabilities to detach sea lice from fish and/or mixtures thereof.

The sea lice copepodites attachment inhibiting semiochemical compositions described herein may be formulated on a chemical carrier with the provisos that: retains the bioactive structure of synthetic palmitoleic acid, salts, isomers, and/or structural analogs thereof that retain the semiochemical capabilities of driving sea lice off fish, and/or mixtures thereof. Such carrier molecules include crown compounds such as crown ethers, liposomes, nanoparticles, microparticles and carrier proteins.

Any kind of liposome can be used to entrap the semiochemical compositions disclosed herein. Liposomes can be made using any natural or synthetic phospholipid such as phosphoglycerides and sphingolipids. Natural phospholipids such as Phosphatidylcholine (PC), Phosphatidylethanolamine (PE) and phosphatidylserine can be used. Synthetic phospholipids that can be used include dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine. Depending on the application, cholesterol may be incorporated into the liposomes. Cholesterol can be incorporated at concentrations varying between 1: 1 or even 2: 1 molar ratios of cholesterol to PC.

Liposomes may be unilamellar vesicles (unilamellar vesicles) or multilamellar vesicles (multiamellar vesicles). Liposomes can also be cross-linked.

Liposomes of the invention can be prepared by methods known in the art using passive loading techniques or active loading techniques. Examples of the mechanical dispersion method include a lipid membrane hydration method, a microemulsion method, an ultrasonic treatment, a French press method, a film extrusion method, a dry reconstituted liposome method, and a freeze-thaw liposome method. The solvent dispersion method comprises ethanol injection, ether injection, and double emulsionVesicles, reverse phase evaporation vesicles, and stable multilamellar vesicles (stable pluralamellar vesicles). For liposome preparation, detergents such as cholate and triton may also be used

And removing the detergent by dialysis, dilution or column chromatography.

In addition, nanoparticles may also be used to deliver semiochemical compositions as described herein. The size of these particles is less than or equal to 100 nm. They can be made of the following materials: natural materials or derivatives, dendrimers (dendrimers), fullerenes, polymers, silica, albumin, gold, hydrogels, and other materials known in the art. Examples of natural materials used to make nanoparticles include chitosan, dextran, gelatin, alginate, and starch. Various polymers that may be used in the nanoparticles of the present invention include polylactic acid, poly (cyano) acrylate, polyvinylamine, block copolymers, polycaprolactone, and poly (lactic-co-glycolic acid) (PLGA).

The nanoparticles may be coated with various materials, such as dextran coatings, enteric coatings, polymer coatings, gold coatings, polyethylene glycol (PEG) coatings, and carbohydrate coatings.

Nanoparticles can be prepared using different methods, such as milling, pyrolysis (pyrolysis), using thermal plasma methods, gas phase techniques, multiple emulsion-solvent evaporation methods, gas flow focusing, electrospray, fluid nanoprecipitation methods, emulsion diffusion-evaporation methods, modified phase inversion/solvent diffusion methods, or sol-gel methods. These methods are described in the literature and are well known to those skilled in the art.

The fine particles are particles having a size of 0.1 to 100 μ M. They can be made of natural polymers or synthetic polymers using materials similar to those of the nanoparticles. Thus, the following are some of the materials used to make the microparticles: cellulose, starch, lysophosphatidylcholine, poly (lactic acid), phosphorylcholine, poly (DL-lactide-co-glycolide), alginate-spermine, polyamino acids, polyphosphazene, albumin, dextran, Eudragit S100, Eudragit L100, gelatin and 3- (triethoxysilyl) propyl-terminated polydimethylsiloxane. The microparticles of the present invention may also be grafted with other materials. As an example, polymethyl methacrylate or polyacrylate grafted starch microparticles, or silicone grafted starch microparticles are used.

Furthermore, the microparticles can also be coated with the same coatings as those described above for the nanoparticles: namely a dextran coating, an enteric coating, a polymer coating, a gold coating, a Eudragit S100 coating, a PEG coating and a carbohydrate coating.

In formulating microparticles, several methods can be used, such as spray-drying, emulsion/evaporation, double emulsion/evaporation, salting out, solvent displacement/precipitation, low temperature preparation (cryo-precipitation) and oil-in-oil emulsion/solvent evaporation. These and other methods are described in the following literature (see, e.g., Kendall et al, eur.j.pharm., Sci 37, 284-290(2009)) and are known in the art.

The semiochemical compositions as described herein may be in the form of: powders, tablets, pills, capsules, granules, granulated particles, dry tablets or other forms suitable for use. Furthermore, it may be in the form of a sustained release formulation disposed in a micelle or microencapsulated. The semiochemical compositions may also be lyophilized.

In one embodiment, the semiochemical compositions as described herein are formulated into non-toxic water dispersible tablets using chemical formulations of polymers known in the art (such as Eudragit, ethylcellulose, microcrystalline cellulose, talc and magnesium stearate). The polymer is mixed with the semiochemical composition and then compressed using a tablet press. The size of the tablets may vary depending on the size of the area to be treated and the number of fish.

Additional carriers that may be added to the formulation include: glucose, lactose, mannose, gum arabic, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silicon dioxide, potato starch, urea, short chain fatty acids, medium chain triglycerides, dextran, fructooligosaccharides (oligofructans) and other carriers suitable for preparing formulations, in solid, semi-solid or liquid form. In addition, auxiliary stabilizers, thickeners or colorants can be used, for example as stabilizers and drying agents, such as for example, acetonose (triulose).

The semiochemical compositions described herein may be diluted in various solutions as described below and may be used in various liquid forms.

Another aspect of the invention is a solution containing a sea lice copepodites attachment inhibiting semiochemical composition comprising a synthesized palmitoleic acid or salts, derivatives, isomers and/or structural analogs thereof that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof.

In another aspect, a solution containing a sea lice copepodites attachment inhibiting semiochemical composition comprising a synthesized palmitoleic acid, or salts, derivatives, isomers and/or structural analogs thereof that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof and a non-toxic filler or enhancer composition forms part of the present invention.

The solution may comprise between about 0.1ppm to about 10ppm, or between about 0.6ppm to about 6ppm, or between about 1ppm to about 5ppm, or between about 0.05ppm to about 20ppm of synthetic palmitoleic acid, or salts, derivatives, isomers, and/or structural analogs thereof, which retain the semiochemical capabilities of driving sea lice off of fish, and/or mixtures thereof. A non-toxic filler or reinforcing agent composition may be added to the solution.

In another aspect, a solution comprising a sea lice copepodites attachment inhibiting semiochemical comprising between about 0.6ppm and about 6ppm of a synthesized palmitoleic acid or salts, derivatives, isomers and/or structural analogs thereof that maintain their semiochemical capabilities to detach sea lice from fish and/or mixtures thereof.

The solution may be made by adding a solvent to a solution of synthetic palmitoleic acid, or salts, derivatives, isomers, and/or structural analogs thereof, which retain their semiochemical capabilities, and/or mixtures thereof. Examples of the solvent include an alkaline solution, ethanol (ethyl alcohol), ethanol (ethanol), ethyl acetate, dimethylformamide, dimethyl sulfoxide, physiological saline, and the like.

The solution may be in the form of a spray, aerosol, underwater diffuser, slow release matrix, injectable, and in the form of drops. It can be added to water as a bath, or it can be placed in the fish food, or it can be applied to the fish, or it can be injected into the fish. Thus, the invention includes oral, topical and injectable treatments. In addition, the present invention also includes placing a semiochemical composition as described herein in a fish environment.

Fish attractants such as cheese, corn kernels, salt shrimp, lobster (crawfish), etc. may be added to the composition or solution as described herein.

Another aspect of the invention is a method of repelling sea lice from fish, said method comprising administering to the fish a semiochemical composition or a semiochemical solution comprising a sea lice copepodites attachment inhibiting semiochemical comprising a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof that maintain their semiochemical capabilities to repel sea lice from fish and/or mixtures thereof and an acceptable vehicle.

Another aspect of the invention is a method of repelling sea lice from fish, said method comprising administering to the fish a semiochemical composition or semiochemical solution comprising a sea lice copepodites attachment inhibiting semiochemical comprising a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs thereof that maintain their semiochemical capabilities to repel sea lice from fish and/or mixtures thereof and a non-toxic filler or enhancer composition and an acceptable vehicle.

Another aspect of the invention is a method of repelling sea lice from fish comprising administering to the fish a semiochemical composition or a semiochemical solution comprising a sea lice copepodites attachment inhibiting semiochemical comprising from about 0.1ppm to about 10ppm, or from about 0.6ppm to about 6ppm, or from about 1ppm to about 5ppm, or from about 0.05ppm to about 20ppm of a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs that maintain their semiochemical capabilities to repel sea lice from fish and/or mixtures thereof and an acceptable vehicle. In addition, non-toxic filler or reinforcing agent compositions may also be added to the composition.

In this method, the semiochemical composition or semiochemical solution is applied for 45 minutes to 2 hours, or 10 minutes to 5 hours, or 20 minutes to 3 hours. The time period depends on how the semiochemical composition as described herein or the semiochemical solution as described herein is formulated.

Another aspect of the invention is a semiochemical composition as described herein or a semiochemical solution as described herein for use in inhibiting the attachment of sea lice to fish.

The semiochemical composition or semiochemical solution for use in inhibiting the attachment of sea lice to fish wherein said semiochemical composition or semiochemical solution comprises between about 0.1ppm and about 10ppm, or between about 0.6ppm and about 6ppm, or between about 1ppm and about 5ppm, or between about 0.05ppm and about 20ppm of a synthesized palmitoleic acid, salts thereof, derivatives thereof, isomers thereof and/or structural analogs that maintain the semiochemical capabilities thereof to detach sea lice from fish and/or mixtures thereof.

The semiochemical compositions and solutions described herein comprise a fatty acid. Fatty acids are available in solid form from various companies. In addition, they may also be chemically synthesized or enzymatically synthesized by methods known in the art. However, because it is difficult to dissolve the fatty acid, the fatty acid is typically added to the solvent under constant stirring and at a temperature between about 37 ℃ and about 38 ℃ (more preferably about 38 ℃).

Once obtained, the compositions of the present invention can be tested for their efficacy in inhibiting sea lice.

The invention will now be illustrated by the following description of examples, which, of course, are not intended to limit the invention in any way. Further, the characteristics of the invention will become clearer from the following observations, which, of course, are intended to illustrate the scope of the invention only and do not limit it in any way.

Examples

Example 1 chemical identification of putative semiochemicals

60 salmonid fish (36 salmon (salmon) and 24 rainbow trout) were sampled for topographic mucus and blood analysis. For the same purpose, a complementary population of 12 salmonids (complementary population) was used for whole-body mucus sampling, while 12 non-salmonids belonging to the codaceae family were also sampled. The codaceae population (population) comprises the following genera (genus) and species (species):

-atlantic pollack (Gadus morhua): 5 fish

-pollack (pollacius virens): 10 fish

-pollack (pollacius): 2 fish

Haddock (Melanogrammus aeglefnus): 1 fish

All samples (including blood samples and blood smears) were chemically analyzed. Blood samples were analyzed for protein, cortisol plasma levels and volatile compounds, while blood smears were analyzed for heterophilic cell/lymphocyte ratios (HLR) and leukocytes. Half of the mucus samples were kept at-18 ℃ for immunological evaluation and protein identification, while the other half was used for identification of volatile compounds.

Blood smears were stained according to the May-Grf ü nwald-Giemsa method for white blood cell count, and then the heterophilic cell/lymphocyte ratio (HLR) was calculated. Basically, the method comprises: blood smears were fixed in methanol for 15 minutes, stained with May-Grunwald for 5 minutes, stained in Giemsa for 10 minutes, washed in buffer at pH 6.8, dehydrated twice in acetone, washed three times in xylene, and counted to 60 cells in total for leukocytes, heterophilic cells, and lymphocytes.

Using a feed from

Life sciences Enzyme Linked Immunosorbent (ELISA) assay, blood samples were tested to assess cortisol plasma levels.

First, volatile compounds are extracted by using different solvents for selective water or fat solubility, such as dichloromethane. This extract was analyzed by GC-GC/MS method. The chromatograms and their variations were further analyzed.

The only indicator considered so far is the plasma concentration of cortisol. The measured values are comparable to values published in the literature. Nevertheless, some abnormal values were obtained, in particular from salmon (salmon).

In salmon (salmon), fish carrying sea lice have no significant difference between them compared to those without parasites. Both groups showed high concentrations of plasma cortisol of about 100,000pg/ml, but both samples were obtained from slaughtered fish just prior to sampling. In contrast, in rainbow trout, infested fish had higher plasma cortisol concentrations (78,490.55pg/ml) than non-infested fish (61,408.75 pg/ml).

The absence of differences in salmon (salmon) may be associated with slaughtering the fish prior to blood sampling.

Blood and mucus samples were extracted using dichloromethane, and then subjected to gas chromatography/mass spectrometry (GC/MS) using a turbo mass spectrometer manufactured by Perkin Elmer. Detection was achieved using bombardment with energy (EI +) at 70eV at 180 ℃. A JW column DB5(id 0.25 mm; 25 μm film; split at 1/20 and split/no split at 45 seconds) with a length of 30m was used.

To confirm the structure of a specific molecule obtained from GC/MS analysis, positive chemical ionization (CI +) in methane was performed to visualize the molecular peak (molecular mass). Such methods are known in the art.

The results are analyzed using a database to obtain the most likely profile. Databases having such data are well known in the art.

Five semiochemicals of possible interest were identified. These semiochemicals are described below.

Putative heterologous pheromone from the pollock (Pollacius) (PASA: pollock anti sea lice heterologous pheromone)

The secretion is obtained from pollack (pollock) (pollack (pollacius virens)). It is an association of volatile compounds that is not present in salmonids. This secretion is of interest, since populations of these fish appear to be present in large numbers in the boxes in which salmonids (salmonids) are grown. Furthermore, these wild fish are not attacked by sea lice, although they consume salmon food, most of which spit back salmon particles after capture, and volatile compounds already present in food and salmonid skin mucus are present in their skin mucus.

Putative allo-pheromones from Healthy salmonids (HRSA1 and HRSA 2: Healthy stable Salmonid allo-pheromone 1 and Healthy stable Salmonid allo-pheromone 2)

In the skin mucus of non-infested salmon and rainbow trout, associations of volatile compounds are found that can help the copepodites avoid colonizing the fish with an effective immune system. Furthermore, by comparing this association of compounds with the analysis of non-infested salmon or trout (which live with infested fish), it is possible to subdivide this association of compounds into two subgroups: HRSA1 and HRSA 2.

Putative interspecific hormones from infesting salmonids (VSSK: easily dangerous Salmonid sea lice interspecific hormones (Vulnerable Salmonid Sealice Kairomonon))

In the skin mucus of infected salmon and rainbow trout, associations of volatile compounds are found which can help the copepodites to select fish which have an impaired immune system and make them vulnerable to parasites. This secretion must be distinguished from another chemical pattern added in fish carrying sea lice.

Putative sea lice attractant pheromone (LHSP: Lepeoptheirus Host Signalling Phero)

The secretion may be released by the lice (after development of the lice on the host), or the secretion may be fish-derived secretion induced by sea lice.

For all screening tests described below, the solutions have been blinded (bound) and the solution for the putative xenohormone was designated A, B, D, E, F and the solution for the putative interspecies hormone was designated M, N, P, Q, R, S. In the following examples, these solutions are described.

Example 2 screening of putative semiochemicals

In assessing the effect of putative semiochemicals, the Smiley Chamber test (FIGS. 2 and 3) was used. This is an experiment adapted from a daily olfactometer.

A set of four 600ml beakers and four 2m long tubes for each new semiochemical were partitioned. The Smiley Chamber and copepods cylinders were washed with warm flowing fresh water for 30 seconds, with detergent for 30 seconds (with the warm fresh water flowing), let warm fresh water flow for another 30 seconds, empty the compartment (Chamber) and spray 2-propanol in the compartment and concentrate on the tube holder and onto the silicone, let warm fresh water flow for another 30 seconds, then let cold water from the water distributor flow, and finally add seawater. This cleaning procedure may be repeated several times, if desired.

Then, three beakers were filled with 300-500 ml of seawater. The remaining beaker was filled with 6ppm semiochemical. The Smiley Chamber was filled with 300ml of seawater, or just enough seawater to cover the tubes. The pump speed was set to 20RPM (5.49 ml/min). The same set-up was done for the tubes on the control and test sides of the compartment and on the beaker.

The test was started by placing the copepoda cylinder between two outlet pipes and then pouring the copepoda into the cylinder while applying pressure on the cylinder by hand. A check was made to ensure that the copepods did not escape the cylinder. A styrofoam wall was placed around the Smiley Chamber and then two lamps placed above the compartment were turned on. Then, the pump was turned on to start timing. After 38 seconds, substance a, containing 30% palmitoleic acid, 20% C13 tridecanal and 50% oleyl alcohol, and substance B, containing 50% palmitoleic acid and 50% squalene, began to enter the compartment. After 3 minutes 30 seconds, the column with the copepodites was slowly raised straight up and the camera was placed on the lid. Photographs were taken at T0, T3min, T6min, T9min and T10 min. After 10 minutes from the end of the experiment, the pump was turned off. The camera, cover and styrofoam wall were removed and then a visual impression of the difference in activity between the control side and the test side was observed, as well as a visually rough count of the copepodites. The data were analyzed to see if the responding copepodites moved in one direction.

Observation experiments with stain showed: after 6 minutes, there was a reflux (reflux) from side to side, resulting in a mixing of the two channels. For this reason, the main parameter evaluated is the picture at T6 min.

The putative heterologous pheromone was tested for competition with conditioned water according to the experimental protocol. The conditioned water is composed of seawater mixed with salmon mucus. To avoid damage to the fish or contamination of the sample with any blood, mucus was obtained by placing 500g of salmon in a disposable plastic bag for 15 seconds. The mucus adhered to the wall of the plastic bag and was then mixed with 600ml of seawater. The conditioned water was stored at +4 ℃ before use in the test.

Three putative interspecifics (E containing 30% palmitoleic acid, 30% oleic acid, and 40% squalene, Q containing 30% palmitoleic acid, 30% oleic acid, and 40% palmitic acid, and R containing 30% lauric acid, 30% palmitic acid, and 40% oleic acid) and 2 putative xenopheromones (a containing 30% palmitoleic acid, 20% C13 tridecanal, and 50% oleyl alcohol, and B containing 50% palmitoleic acid and 50% squalene) were examined. Tests using conditioned water were used to identify the appropriate concentrations required for the tests.

In the case of 3 species of test interspecies hormones, E, containing 30% palmitoleic acid, 30% oleic acid and 40% squalene, is the most effective compound, showing a very pronounced attractant effect and causing significant agitation in the copepods. Considering that the reference attractant isophorone was used as a control, a solution E containing 30% palmitoleic acid, 30% oleic acid and 40% squalene showed very comparable efficacy. Q, containing 30% palmitoleic acid, 30% oleic acid, and 40% palmitic acid, although multifunctional, did not have comparable effects.

E containing 30% palmitoleic acid, 30% oleic acid and 40% squalene, the putative interspecific hormone obtained from susceptible salmon seems to be the interesting one, whose efficacy must be confirmed in further experiments.

Code E as described above corresponds to the putative interspecies hormone group VCCMIS (volatile compounds from cutaneous mucus invading salmonids).

Allogenic pheromone a (containing 30% palmitoleic acid, 20% C13 tridecanal and 50% oleyl alcohol) and allogenic pheromone B (containing 50% palmitoleic acid and 50% squalene), identified in secretions of non-invasive salmon and trout living in infected cages, were tested for their competition with conditioned water. In 4 replicates, B did not show any visible effect. Conversely, a as described above shows several interesting effects. 10 replicates were performed, of which only 5 provided useful data. A appears to inhibit the attracting effect of the conditioned water. As mentioned above, a belongs to the group of putative heterologous pheromones HRSR (released by healthy stable salmonids).

Example 3 screening by Linear olfactory Meter

The aim of this test is to obtain more precise results with greater precision in the copepodites counting. Since the copepodites appeared to have limited mobility, the olfactometer was simplified using a small device divided into 3 compartments (fig. 4). The system first verifies the symmetry of those channels carrying semiochemicals (one channel with a reference using ethanol as solvent; one channel with a test mixture). Another improvement in the protocol was an improvement in the counting method to ensure that the number of copepods introduced in the olfactometer and counted in the respective compartment was accurately counted.

To count the copepods, they were first fished out of the housing, then divided into drops and placed in wells of an ELISA-type plastic plate. Stereomicroscopes are used to count the copepods so that the exact number of copepods is known and input to the olfactometer. The linear olfactometer is divided into 3 internal compartments: left and right branches, and a central region, plus an outer outflow region (where the copepods were collected in a petri dish and filtered to count the copepods).

30 copepodosomal larvae were added to the main branch of the olfactometer, where they were faced with a constant flow (0.84ml/s) at a temperature of 8-12 ℃. This stream was divided into 2 sub-streams, each from a 250mL bottle. From one subpool stream, the copepodites received only the solvent (ethanol) for the test product, and from the other subpool stream, the copepodites received the putative semiochemical mixture at a concentration of 10 ppm. The test was run for ten (10) minutes. After this period of time, the flow of liquid was stopped and surgical forceps were applied to the tube on the olfactometer to block the water contained in the 4 zones. The water was examined by stereomicroscope to count the copepodites (fig. 5).

3 putative xenobiotic cocktails (A, B, D) and 4 putative interspecies hormones (E, Q, R, S) were tested. The heterologous pheromone mixture comprises the following compositions:

a: contains 30% of palmitoleic acid, 20% of C13 tridecanal and 50% of oleyl alcohol.

B: contains 50% palmitoleic acid and 50% squalene.

D: contains 60% oleyl alcohol and 40% squalene.

The interspecies hormone blend comprises the following compositions:

e: contains 30% palmitoleic acid, 30% oleic acid, and 40% squalene.

Q: contains 30% palmitoleic acid, 30% oleic acid, and 40% palmitic acid.

R: contains 30% lauric acid, 30% palmitic acid and 40% oleic acid.

S: contains 40% lauric acid, 40% myristic acid and 20% oleic acid.

The results are shown in tables 1 to 6 below. Statistical analysis was calculated by Wilcoxon signed rank (Wilcoxon signed rank).

TABLE 1 heterologous pheromone A

In the absence of data from number 6, the data was recalculated, as described below in table 2.

TABLE 2 heterologous pheromone A (nothing #6)

The following figures show: due to the xenobiotic pheromone a, the average number of copepods was significantly higher in the placebo arm than in the treatment arm.

TABLE 3 interhormone E

TABLE 4 interhormone Q

TABLE 5 interhormone R

TABLE 6 interhormone S

Among all the mixtures used in this test, the only one that provides truly interesting results is a. This solution can be considered as a putative heterologous pheromone mixture. In contrast, the test using E resulted in some conflicting results, products (did not provide significant attractant effect). The contradiction with the previous examination may be a consequence of the lack of precision in the Smiley Chamber system.

Example 4: evaluation of putative semiochemicals in infestation tests

In view of the results of the previous tests, attention was focused on putative semiochemicals A, B, E, F and P, which were actively selected or not tested in the tests described in the foregoing examples 1-3. Based on these results, an attempt will be made to identify the specific signal that caused the detachment of the copepodites.

The following are compounds described in semiochemicals A, B, E, F and P:

B: contains 50% palmitoleic acid and 50% squalene.

E: contains 30% palmitoleic acid, 30% oleic acid, and 40% squalene.

F: contains 50% of squalene, 30% of C13 tridecanal and 30% of oleic acid.

P: contains 17% myristic acid, 17% palmitic acid, 56% palmitoleic acid, and 10% oleic acid.

The test exposed juvenile salmon (smolt) to a high density group of copepodites for 45 minutes in a test apparatus comprising four 23cm diameter tanks with outflow openings (outflow). The inspection device is protected with a tent which does not allow any shadows or uncontrolled light around the fish. Light may alter the behavior of the photic copepodites. Each tank receives parr for 45min (fig. 6).

First, the copepods were fished out of their incubator tanks and counted on a stereomicroscope. Four infested doses of copepods (each containing 60 copepods) were prepared for each test run. Smolt were placed in a tank containing treatment solution (or placebo) at a concentration of 6ppm in the active. The bath lasted for 10 minutes.

Smolt were then placed in a tank, where smolt was kept at 3ppm for the same treatment (product or placebo) as used in the previous water bath, to start the test. The aim of this process is to prevent any significant reduction in the concentration of the active product on their skin.

During the first 10 minutes, the outflow was opened and the tent was closed. At that point, each tank is refilled to reach the initial volume of oxygenated liquid (refilled with the same solution, product or placebo). The outflow was then closed for 5 minutes and the infesting dose was added to the tank. Then, the outflow port was opened, and the opened state was maintained for 30 minutes, and refilling was performed every 10 minutes (tent was not opened).

After 45 minutes of exposure to the copepodites, toxic doses of anesthetic are administered at a concentration 10 times the normal dose (30-40 mg/L)

Smolt was euthanized.

Dead fish were placed in plastic bags where they were wiped and rinsed with seawater. As shown in fig. 7, the skin of the fish was scraped with a metal spoon to remove all the copepodites that may be attached to the fish.

Each fish was washed so that all copepods and shellfish were detached from the skin. The liquid collected from the washing liquid was filtered to count the parasites. The copepods were counted on the filter to know the number of copepods on the external body of the fish. Then, the gills were dissected to count the number of copepods on the gills.

The first stage of the test is to compare the number of copepods, both physically and gill, between replicates of the test. The purpose of this stage is to verify the verification and measure its standard deviation.

The second phase of the test was a comparison of the number of body and gill copepods (abbreviated as cop in the following table) between treated and reference parrs, and as a control, the reference parrs were bathed in five solutions of seawater plus ethanol only. The results are shown in the following table.

TABLE 7 Reference/Control

TABLE 8 semiochemical A

TABLE 9 Chemofenpenin B

TABLE 10 semiochemical E

TABLE 11 semiochemical F

TABLE 12 semiochemical P

The reproducibility of the test seems to be very interesting, wherein the number of copepods on the fish seems to be constant. According to the literature (Tully et al, 2002), this infestation is low and very likely involves very high concentrations of copepodites in this test.

These data did not have any significant effect on any of the test solutions. Observing the results obtained with a as described above, it shows: the solution did not show the same effect in this test compared to the previous test. A as described above was not able to inhibit the attachment of the copepodites to parr. Despite this lack of significance, the number of body copepodites on parr treated with a as described above was somehow low compared to the reference or compared to other putative xenohormones. Interestingly, all of those putative mixtures contained a common compound, palmitoleic acid.

Example 5-repeat example 4 using palmitoleic acid

The same test as in example 4 was repeated using palmitoleic acid. The results are shown in Table 13 below.

TABLE 13 palmitoleic acid

Palmitoleic acid provides an important infection reduction.

This test presents a possible "body infection" problem revealed by such experimental infections, in contrast to the data of Bron et al (1993) and Tully et al (2002). The copepodites are affected by sudden accelerations and tend to attach to moving bodies (Heuch and Karlsen, 1997). In this example, a population of high density copepodites continuously surrounds the fish. It is reasonable to suspect that even in the presence of an effective xenobiotic, it can lead to multiple successive attachment-detachment of the copepodites. Thus, at the end of the test, there are always some copepods that have just adhered to the body and that have no time to detach when harvested on dead fish. This hypothesis reveals that any attempt to validate a potential heterologous pheromone product risks false negative results.

Example 6 validation of the heterologous pheromone palmitoleic acid

The best way to confirm this hypothesis (no time to detach the copepod before it is harvested on dead fish) is to perform the same test in three arm protocols: two identical branches as preliminary experimental protocol (reference branch and treatment branch), and a novel branch comprising a negative reference, were carried out by the following fish: this species is not a natural host for Phlebopus scabiosus (Lepeopthheirus).

In this example, the synthetic palmitoleic acid was tested according to the exact same experimental protocol, except that the following 3 groups of fish were used: (1) positive reference: untreated smolt; (2) negative reference: juvenile cod (atlantic pollack (Gadus morhua)) of comparable size; and (3) treatment groups: smolt that received palmitoleic acid.

The same experiment was performed as described in example 4. The results are set forth in table 14 and table 15 below.

TABLE 14

Watch 15

The boxplot results are shown in FIG. 8. The results were statistically analyzed, as shown in FIGS. 9 to 11. Levees test was performed, which is a reasoning statistic used to evaluate homogeneity of variance (Equality of varians) of variables calculated for more than two groups. The results are shown in Table 16 below.

TABLE 16

The distribution of group 3 was normal (normal).

Tukey's HSD assay was performed and the results are shown in Table 17 below.

TABLE 17

These results show that: there was a highly significant difference between control salmon and control cod, a highly significant difference between control salmon and treated salmon, and no significant difference between treated salmon and control cod.

Discussion and conclusions:

the hypothesis of "passive body infestation" in examples 4-6 was verified. The copepodites attach to cod, a fish that is not a natural host for Lepeoptheirus. Infections on gills are also passive infections, which are highly correlated with high density of copepods in experimental infections.

The heterologous pheromone palmitoleic acid induces a significant decrease in the number of copepodites attached to the salmon body. Salmon treated with synthetic palmitoleic acid is infested in a manner comparable to cod, a natural, non-infested species of lepeoptheirus salmonis.

Example 7-efficacy of isomers of semiochemicals to inhibit sea lice copepodites attachment as inhibitors of infestation of Atlantic SALMON (Atlantic SALMON/Salmo salar) by copending copepodites

The purpose of this example was to examine the efficacy of isomers of the semiochemical (SCAIS) that inhibit sea lice copepodites attachment as inhibitors of the infestation of Atlantic Salmon (Atlantic salon/Salmonella salmonis) by the copepodites of the common scab (Lepoephheir salmonis).

The isomer of semiochemical (SCAIS) used in this example to inhibit sea lice copepodites attachment is trans-9-hexadecenoic acid.

The test was carried out by using 4 rounds of 4 fish, 8 of which were treated with trans-9-hexadecenoic acid, while the other 8 were used as controls. For each round, 4 fish were tested; i.e. 2 treated fish and 2 controls.

To be included in the study, parr must be between 70g and 150g in weight and the copepodites must be able to swim actively. If juvenile salmonids are diseased (scale defects, fin damage, cataracts, and/or abnormal swimming), they are not used in the study. If the copepods were immobile after stimulation, they were removed from the study.

For each round, 4 fish were caught and introduced into 4 2-L flat bottom beakers (to which 1.75L of seawater was added). The treated fish had 6ppm trans-9-hexadecenoic acid in their seawater, while only seawater was used in the control. 0.52ml of the treated or control was injected directly into 1.75L of seawater. 4 fish were bathed in this solution for 10 minutes.

The fish were then transferred to another 3.5L beaker (to which was added 3.5L of seawater) treated with 3ppm of trans-9-hexadecenoic acid, or seawater only control. 0.52ml of the treated or control was injected directly into 3.5L of seawater. The beaker was equipped with a valve to empty 0.875L from the beaker. When fish are introduced into the beaker, the valve is opened. After 10 minutes of valve opening, 0.875L of treated trans-9-hexadecenoic acid or 0.875L of control seawater was added to the beaker. After 10 minutes (20 minutes after the fish were introduced into the beaker), the valve was closed and the fish were added to each flat-bottomed beaker in a proportion of 60 copepods per fish. After 5 minutes (25 minutes after the fish were introduced into the beaker), the valve was opened. Later 10 minutes (35 minutes after the fish were introduced into the beakers), each beaker was supplied with 0.875L of trans-9-hexadecenoic acid or 0.875L of control seawater. After 10 minutes (45 minutes after the fish were introduced into the beakers), 0.875L of trans-9-hexadecenoic acid or 0.875L of control seawater was added to each beaker. After 10 minutes (55 minutes after introducing the fish into the beakers), 0.875L of trans-9-hexadecenoic acid or 0.875L of control seawater was added to each beaker.

Then, in order to kill the fish with an excess of anaesthetic product, 2ml of

Injection into each flat-bottomed beaker. The fish are then introduced into the coded plastic bag. In the gill chamber, the fish was held with forceps and scrubbed three times on different parts of the fish. The top of the fish is first scrubbed in the plastic bag and then rinsed, followed by scrubbing the bottom of the fish in the plastic bag and then rinsing, and then scrubbing and rinsing the entire fish in the plastic bag. The fish were then removed from the plastic bag and weighed. The contents of the plastic bag were then emptied onto filters, and the number of copepods was then counted on each filter using a magnifying glass. The process is repeated for other fish and other rounds of fish.

Table 18 shows the number of rounds of fish tested and whether they were treated with trans-9-hexadecenoic acid or with control seawater.

Watch 18

Wheel	Left side of	Center left	Center right	Right side
						1	SCAIS^{Different from each other}	SCAIS^{Different from each other}	Control	Control
2	Control	SCAIS^{Different from each other}	SCAIS^{Different from each other}	Control
					3	Control	Control	SCAIS^{Different from each other}	SCAIS^{Different from each other}
4	SCAIS^{Different from each other}	Control	Control	SCAIS^{Different from each other}

Among them, SCAIS in Table 18^{Different from each other}Refers to trans-9-hexadecenoic acid.

Table 19 is the results obtained from this example.

Watch 19

Among them, SCAIS in Table 19^{Different from each other}Refers to trans-9-hexadecenoic acid.

The experiment was repeated using stock fish, with standard or experimental defects excluded, until 8 treated and 8 control fish were obtained (results). Outliers (atypical values) are deleted from the data if they are characterized as paradoxical results. If the centralization (centering) and reduction of the data is higher than the absolute value of 3, it is considered an outlier. No atypical values were found in this study.

Data analysis was performed using 9.4SAS software (2002-. All data were tested using a univariate program in the SAS 9.4 software, using residual diagnostics plots (residual diagnostics plots) to demonstrate the assumption of deviation from normality. Comparisons between control and treated groups (based on body mass and number of attached copepods) were made according to normal and variance using the student test using the t test program in the SAS94 software, or the Wilcoxon two-sample test using the nparway program. The homogeneity of the variance (homogeneity) was verified using a fish test with the t test program. The significance threshold (significance threshold) is typically set to 5%.

Watch 20

Among them, SCAI in Table 20^{Different from each other}Refers to trans-9-hexadecenoic acid, and Nb refers to quantity.

The results of the normal test were as described in Table 21 below.

TABLE 21

The results of the normal test were performed and the control results are described in table 22 below.

TABLE 22

All the tests used concluded a normal body mass for the fish tested against trans-9-hexadecenoic acid and the control.

The results of the fisher test for homogeneity of variance are shown in table 23 below.

TABLE 23

For "body mass," the variance between treatment groups is homogeneous.

The results of using Student's t-test are shown in Table 24 below.

Watch 24

Salmon were homogenous between treatment groups according to "body mass". (p as 0.4027)

Conclusion

For "body mass", the Atlantic salmon were homogeneous between the test groups. "Trans-9-hexadecenoic acid (SCAIS)^{Different from each other}) "has a significant effect on the number of copepodites attached to Atlantic salmon, whereas they are attached to trans-9-hexadecenoic acid (SCAIS)^{Different from each other}) The number of copepods in the treated salmon was low.

EXAMPLE 8 preparation of a tablet of semiochemical for inhibiting adhesion of sea lice copepodites

A water-dispersible tablet containing a semiochemical that inhibits adhesion of sea lice copepodites was prepared as follows. 132g of the sea lice copepodites attachment inhibiting semiochemical was blended with 150g of Eudragit RL, 200g of ethyl acetate and 110g of microcrystalline cellulose. 25% magnesium stearate and 5% talc were blended together and then added to the initial formulation containing semiochemicals. The mixture was compressed using a station rotary tablet press (with a flat punch of 8mm diameter).

EXAMPLE 9 efficacy of a tablet of semiochemical to inhibit adhesion of sea lice copepodites

The purpose of this example was to evaluate the efficacy of the semiochemical (SCAIS, which is continuously released by water-dispersible tablets) that inhibits adhesion of sea lice copepodites on the infestation of Atlantic salmon parr (Atlantic salmon) by the Salmonella salmonides copeus copepodites.

The sea lice copepodites attachment inhibiting semiochemical (SCAIS) used in this example was cis-9-hexadecenoic acid (palmitoleic acid).

In this study, a weight of approximately 90g was used during the parr period and produced from

(Daugstad 6392 Vikebukt, Norway) 72 Atlantic salmon (Salmo salar). 2400 copepodites used were from species Lepeoptheirus salmonis (Lepeoptheirus salmonis) produced from

(Bergen, norway).

Parr were excluded from the data under study if they were swimming on their backs during the trial. The infestation test is measured at 1 hour, 24 hours, 72 hours and 120 hours after the treatment has been carried out. Blood tests were performed at 0 hours, 1 hour, 24 hours, 72 hours, and 120 hours after treatment.

Studies were performed in two parallel groups of Atlantic salmon smolt using 40 fish per tank: one group was treated with cis-9-hexadecenoic acid in semiochemical tablets to inhibit sea lice copepodites attachment placed in the middle under a mesh bag (string bag) where water resides in the habitat and the other group was a control using only ethanol.

To obtain a blood sample, 4 fish were individually harvested from each of the two tanks and introduced into a volume of 0.7ml/L

In an anaesthetic bath. After 1 minute, blood samples were collected in the tail vein of the fish using a 25ml syringe with a 0.6mm needle. A drop of blood is placed on a glass slideAbove, a blood smear was performed using a plastic strip. The ratio (H/L) of heterophilic granulocytes (heteophils) to lymphocytes from these blood smears was measured using a Diff-Quickstain (Diff-quick, a modified stain of the Wright-giemsasain stain). The remaining blood was injected into 4ml heparinized tubes and stored in a refrigerator at-18 ℃ until subsequent plasma cortisol analysis. All plasma cortisol was tested by enzyme-linked immunosorbent (ELISA) assay kit, which was derived at 1: 25 (ng.ml)^-1) Diluted blood samples.

The infection test was performed at 1 hour, 24 hours, 72 hours, and 120 hours after the treatment was performed in the following manner. Two fish were individually housed in two tanks (control and test) and introduced into 4 3.5L beaker pans (which were supplemented with 3.5L of water sampled from the original tank of each fish). The flat bottom beaker was equipped with a valve that was able to empty 0.875L of solution when an equal amount of solution was introduced into the beaker. When fish are introduced into the beaker, the valve is opened. After 10 minutes of fish introduction, 0.875L of their respective solutions (test or control) were added to the flat bottom beaker. After 10 minutes (20 minutes after the fish were introduced into the flat-bottomed beaker), the valve was closed. The (proportion of) 60 copepods per fish will be added to each flat-bottomed beaker. After 5 minutes of adding the copepodites (25 minutes after introducing the fish into the flat bottom beaker), the valve was opened. After 10 minutes (35 minutes after the fish were introduced into the flat-bottomed beakers), each flat-bottomed beaker was charged with its respective treatment solution (cis-9-hexadecenoic acid or control). After 10 minutes (45 minutes after the fish were introduced into the flat-bottom beakers), each flat-bottom beaker was charged with its respective treatment solution (cis-9-hexadecenoic acid or control). After 10 minutes (55 minutes after the fish were introduced into the flat-bottomed beakers), each flat-bottomed beaker was charged with its respective treatment solution (cis-9-hexadecenoic acid or control). After 10 minutes (65 minutes after the fish were introduced into the flat-bottomed beaker), 2ml of

Adding into each flat-bottomed beaker to allow excess hemp to pass throughDrunk to kill fish. When the fish die, they are introduced separately into a plastic bag.

The individual fish were kept in plastic bags using forceps, which were introduced into the gill chamber of the fish. Then, scrub the fish 3 times in the plastic bag; i.e., water is used to scrub and rinse the top of the fish, water is used to scrub and rinse the bottom of the fish, and water is used to scrub and rinse the entire fish. The fish were then removed from the plastic bag and weighed. The water contents of the plastic bag were then emptied into a filter to collect the copepods. The number of copepods on the respective filters was counted using a magnifying glass.

The process was repeated for the other 7 rounds of fish.

The experiment was repeated using stock fish with standard or experimental defects excluded. This type of data is not included in the overall result in order to handle missing data and outliers.

Preliminary analysis showed a positive trend in favor of the treated group. The average number of copepodites attached to control parr was 15.4, while the average number attached to the treated group was 10.6. These results show that: semiochemicals (cis-9-hexadecenoic acid) which inhibited sea lice copepodites attachment bound rapidly and effectively to the mucus of fish to provide significant protection in a closed 3.5L tank test of high intensity infestation (60 copepodites per fish).

Isomers of palmitoleic acid (cis-9-hexadecenoic acid) and trans-9-hexadecenoic acid can be considered as SCAIS (semiochemical inhibiting adhesion of sea lice copepodites) responsible for screening for acceptable hosts for salmon lice. Palmitoleic acid (a low molecular weight compound, considered as a metabolite in many species, and well known for its non-toxicity) is a promising option for preventing sea lice infestation in salmon farming.

While the invention has been described in terms of various preferred embodiments, the skilled artisan will appreciate that: various modifications, substitutions, omissions, and changes may be made without departing from the scope thereof. It is therefore intended that the scope of the invention be defined by the scope of the following claims (including equivalents thereof).

Claims

1. Use of synthetic palmitoleic acid, salts thereof, and/or trans-9-hexadecenoic acid, in combination with an acceptable excipient, in the manufacture of a composition for inhibiting sea lice copepodites attachment to fish.

2. Use according to claim 1, wherein the composition comprises 0.1-10 ppm of synthetic palmitoleic acid, its salts and/or trans-9-hexadecenoic acid and acceptable excipients.

3. Use according to claim 1, wherein the composition comprises 0.6-6 ppm of synthetic palmitoleic acid, its salts and/or trans-9-hexadecenoic acid and acceptable excipients.

4. The use according to claim 1, wherein the acceptable excipient is a pharmaceutically acceptable excipient or a veterinarily acceptable excipient.

5. The use according to claim 1, wherein the composition can be in the form of a powder, tablet, pill, capsule, granulate, granular granule, dry tablet, in the form of a sustained release formulation, placed in micelles, liposomes, nanoparticles, microparticles, microencapsulated or lyophilized.

6. Use according to claim 1, wherein the composition is in the form of a solution.

7. The use of claim 6, wherein the solution is formulated as a spray, aerosol, emulsion, suspension, drops placed in an underwater diffuser or in a slow release matrix.

8. Use according to claim 6, wherein the solution can be added to water in which the fish reside, or placed in fish food.

9. The use of claim 6, wherein the solution is administered to the fish orally or by injection.